Growth arrest homeobox gene

ABSTRACT

A novel growth arrest homeobox gene has been discovered and the nucleotide sequences have been determined in both the rat and the human. The expression of the novel homeobox gene inhibits vascular smooth muscle cell growth. The growth arrest homeobox gene hereinafter referred to as the “Gax gene” and its corresponding proteins are useful in the study of vascular smooth muscle cell proliferation and in the treatment of blood vessel diseases that result from excessive smooth muscle cell proliferation, particularly after balloon angioplasty.

BACKGROUND OF THE INVENTION

[0001] The leading cause of death in the United States and in mostdeveloped countries, is atherosclerosis. Atherosclerosis is a diseaseaffecting the large and medium size muscular arteries such as thecoronary or carotid arteries and the large elastic arteries such as theaorta, iliac, and femoral arteries. This disease causes narrowing andcalcification of arteries. The narrowing results from deposits ofsubstances in the blood in combination with proliferating vascularsmooth muscle cells.

[0002] The deposits known as atherosclerotic plaques are comprised oflipoproteins, mainly cholesterol, proliferating vascular smooth musclecells and fibrous tissue, and extra cellular matrix components, whichare secreted by vascular smooth muscle cells. As the plaques grow, theynarrow the lumen of the vessel decreasing arterial blood flow andweakening the effected arteries. The resulting complications potentiallyinclude a complete blockage of the lumen of the artery, with ischemiaand necrosis of the organ supplied by the artery, ulceration andthrombus formation with associated embolism, calcification, andaneurysmal dilation. When atherosclerosis causes occlusion of thecoronary arteries, it leads to myocardial disfunction, ischemia andinfarction and often death. Indeed, 20-25% of deaths in the UnitedStates are attributable to atherosclerotic heart disease.Atherosclerosis also leads to lower extremity gangrene, strokes,mesenteric occlusion, ischemic encephalopathy, and renal failure,depending on the specific vasculature involved. Approximately 50% of alldeaths in the United States can be attributed to atherosclerosis and itscomplications.

[0003] Present treatments for atherosclerosis include drugs and surgery,including ballon angioplasty. As a result of angioplasty, vascularsmooth muscle cells de-differentiate and proliferate and leading toleading to reocclusion of the vessel. These de-differentiated vascularsmooth muscle cells deposit collagen and other matrix substances, thatcontribute to the narrowing of vessel. Vascular cells secrete growthfactors such as platelet derived growth factor, which induces bothchemotaxis and proliferation of vascular smooth muscle cells.

[0004] Many of the present drug therapies treat a predisposing conditionsuch as hyperlipidemia, hypertension, and hypercholesterolemia, in anattempt to slow or halt the progression of the disease. Other drugtherapies are aimed at preventing-platelet aggregation or thecoagulation cascade. Unfortunately, the drug treatments do not reverseexisting conditions.

[0005] Surgical treatments include coronary artery bypass grafting,balloon angioplasty, or vessel endarterectomy which, when successful,bypass or unblock occluded arteries thereby restoring blood flow throughthe artery. The surgical treatments do not halt or reverse theprogression of the disease because they do not affect smooth muscle cellproliferation and secretion of extra cellular matrix components.

[0006] The bypass surgeries, particularly the coronary bypass surgeries,are major, complicated surgeries which involve a significant degree ofrisk. The balloon angioplasty, while also a surgical procedure, is lessrisky. In balloon angioplasty, a catheter having a deflated balloon isinserted into an artery and positioned next to the plaque. The balloonis inflated thereby compressing the plaque against the arterial wall. Asa result, the occlusion is decreased and increased blood flow isrestored. However, the balloon angioplasty injures the arterial wall. Asa result, the underlying vascular smooth muscle cells migrate to theintima, and synthesize and excrete extracellular matrix componentseventually leading to the reocclusion of the vessel. Of the estimated400,000 coronary artery balloon angioplasties performed each year in theUnited States, 40% fail due to reocclusion requiring a repeat procedureor coronary bypass surgery. Bypass surgeries also have a significantrate of failure due to internal hyperplasia, which involves excessiveproliferation of vascular smooth muscle cells at the sites of vascularanastamoses.

[0007] Attempts have been made to prevent reocclusion of vessels afterballoon angioplasties in experimental animals. One approach has been totreat rat carotid arteries with antisense oligonucleotides directedagainst the c-myb gene following balloon angioplastyde-endothelialization. In vascular smooth muscle cells the Theexpression of the c-myb gene is up-regulated during the G1 to Stransition of the cell cycle, and the activation of c-myb expression isrequired for further cell cycle progression. The antisenseoligonucleotides to c-myb blocked smooth muscle cell proliferationfollowing balloon angioplasty. However, the antisense oligonucleotidesare applied in a pleuronic gel to the adventitia, that is, the exterior,rather than the lumen side of the affected vessel. Exposing the theexterior of the vessel requires additional surgery with its attendantrisks, and is therefore not desirable.

[0008] It would be desirable to have a nonsurgical treatment, used inconjunction with balloon angioplasties to reduce vascular smooth musclecell proliferation.

SUMMARY OF THE INVENTION

[0009] A novel growth arrest homeobox gene has been discovered and thenucleotide sequences have been determined in both the rat and the human.The expression of the novel homeobox gene inhibits vascular smoothmuscle cell growth. The growth arrest homeobox gene hereinafter referredto as the “Gax gene” and its corresponding proteins are useful in thestudy of vascular smooth muscle cell proliferation and in the treatmentof blood vessel diseases that result from excessive smooth muscle cellproliferation, particularly after balloon angioplasty.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 is the nucleotide sequence of rat Gax gene with thepredicted amino acid sequence listed below the nucleotide sequence. Thehomeobox is indicated by a box, and the CAX nucleotide repeat, where Xis ether cytosine or guanine, is underlined. A polyadenylation signal isin boldface and italics. Putative consensus sites are indicated asfollows: for phosphorylation by protein kinase C, circles; for cyclicAMP (cAMP)-dependent protein kinase, squares; for casein kinase II,diamonds; and for histone H1 kinase, triangles. Residues which couldpotentially be a target for either cAMP-dependent protein kinase orprotein kinase C are both circled and boxed.

[0011]FIG. 2 is the map of mouse chromosome 12 showing the location ofthe Gax gene;

[0012]FIG. 3 is the nucleotide sequence of human Gax gene with thepredicted amino acid sequence listed below the nucleotide sequence;

[0013]FIG. 4 is a map of human Gax gene showing how the separatelycloned fragments were joined and oriented in the plasmid, BluescriptIISK+;

[0014]FIG. 5A is a northern blot showing Gax RNA levels in vascularsmooth muscle cells in response to 10% fetal calf serum after 4, 24, and48 hours; lane Q is RNA from quiescent cells; GAPDH is ratglyceraldehyde 3-phosphate dehydrogenase;

[0015]FIG. 5B is a northern blot showing Gax RNA levels and Hox 1.3 RNAlevels in vascular smooth muscle cells in response to 10 ng/ml humanplatelet derived growth factor at 0.25, 0.5, 1, 2, and 4 hours, lane Qis RNA from quiescent vascular smooth muscle cells;

[0016]FIG. 6 is a graph of changes in relative Gax mRNA levels invascular smooth muscle cells in response to 10% fetal calf serum and 10mg/ml of the PDGF isoforms; the circles represent PDGF-AA, the squaresrepresent PDGF-BB, the diamonds represent fetal calf serum, and thetriangles represent PDGF-AB;

[0017]FIG. 7 is a graph showing ³H-thymidine uptake in vascular smoothmuscle cells at various times after stimulation with fetal calf serumand PDGF isoforms; the circles represent PDGF-AA, the trianglesrepresent PDGF-AB, the squares represent PDGF-BB, the diamonds representfetal calf serum, and the solid squares represent no mitogen;

[0018]FIG. 8 is a graph showing relative Gax mRNA levels in vascularsmooth muscle cells in response to varying doses of PDGF-AB, representedby triangles, and PDGF-BB, represented by squares;

[0019]FIG. 9 is a graph showing relative Gax mRNA levels in vascularsmooth muscle cells in response to varying doses of fetal calf serum;

[0020]FIG. 10 is a graph showing relative Gax mRNA levels in vascularsmooth muscle cells in response to fetal calf serum withdrawal;

[0021]FIG. 11 is a dose response curve showing % inhibition of growth invascular smooth muscle cells in response to varying doses ofmicroinjected GST-Gax protein;

[0022]FIG. 12 is a graph showing percent inhibition of mitogen inducedDNA synthesis in vascular smooth muscle cells in response to: ras(Leu-61) protein; ras (Leu-61) protein in combination with the GST-Gaxprotein; GST-Gax protein; and the GST;

[0023]FIG. 13 is a graph showing percent inhibition of vascular smoothmuscle cell entry into S phase by microinjected GST-Gax protein overtime and the ³H-thymidine uptake over the same time period;

[0024]FIG. 14 is a graph showing the ratio of the Gax mRNA toglyceraldehyde-3-phosphate dehydrogenase designated “G3” level fromnormal vascular tissue and times following acute blood vessel injury.

DETAILED DESCRIPTION OF THE INVENTION

[0025] A novel gene, the Gax gene, has been discovered, the expressionof which inhibits vascular smooth muscle cell growth. The Gax gene andthe protein it encodes, referred to herein as the “Gax protein” areuseful in the study of vascular smooth muscle cell proliferation and ininhibiting smooth muscle cell proliferation. The inhibition of vascularsmooth muscle cell proliferation, particularly by genetic therapy, isalso useful in the treatment of vascular diseases associated withexcessive smooth muscle cell proliferation.

[0026] Nucleotide sequences, such as the Gax gene or portions therof, ormRNA are administered to the vascular cells, preferably during a balloonangioplasty procedure, to inhibit the proliferation of vascular smoothmuscle cells. The nucleotide sequences are delivered, preferably to theinterior of the vessel wall during balloon angioplasty procedurepreferably by a perforated balloon catheter. Genes are transfered fromvectors into vascular smooth muscle cells in vivo where the genes areexpressed. Suitable vectors and procedures for the transfer ofnucleotides are found in

[0027] Nabel, E. G., et al. “Site-Specific Gene Expression in Vivo byDirect Gene Transfer into the Arterial Wall” (1990) Science Vol. 249,pp. 1285-1288, which is incorporated herein by reference. Specializedperforated balloon catheters which deliver nucleotide sequences tovessel walls employing viral and non-viral vectors are used for deliveryof nucleotide sequences and a description of the catheter's structureand use may be found in Flugelman M. Y., et al. “Low Level In Vivo GeneTransfer Into the Arterial Wall Through a Perforated Balloon Catheter”Circulation, Vol. 85, No. 3, pp. 1110-1117 (March 1992) which isincorporated herein by reference.

[0028] Genetic therapy, preferably by the over expression of the Gaxgene, restores the proliferating vascular smooth muscle cells to a morenormal phenotype, thus preventing or reducing the smooth muscleproliferation that is associated with the formation of the atheromatousplaque and with internal arterial thickening following balloonangioplasty. In addition to preventing or reducing the reocclusion ofthe vessel, such genetic therapy decreases the risks associated withadditional surgeries. Also, the Gax proteins or portions thereof, areadministered to vascular cells preferably employing the perforatedcatheter, to inhibit the proliferation of vascular smooth muscle cells.

[0029] The molecular control of cellular proliferation is not wellunderstood. A class of genes, known as Homeobox genes, encode a class oftranscription factors which are important in embryogenesis, tissuespecific gene expression and cell differentiation. The homeobox genesshare a highly conserved 183 nucletide sequence that is referred to asthe “homeobox”. The homeobox encodes a 61 amino acid helix-turn-helixmotif that binds to adenine and thymine rich gene regulatory sequenceswith high affinity. Several vertebrate homeobox proteins have been shownto be transcription factors required for expression of lineage-specificgenes. The tissue-specific transcription factors bind to DNA and repressor induce groups of subordinate genes. Many, but not all of thesehomeobox genes are located in one of four major clusters known as Hoxclusters, designated Hox-1, Hox-2, Hox-3 and. Hox-4. The Hox genes areexpressed in the developing embryo, in distinct overlapping spatialpatterns along the anterior-posterior axis which parallels the Hox geneorder along the chromosome. Homeobox transcription factors control axialpatterning, cell migration and differentiation in the developing embryoand are involved in the maintenance of tissue specific gene expressionin adult organisms.

[0030] A new homebox gene has been discovered, isolated and sequenced inboth the rat and human. This new gene is a growth arrest specifichomeobox gene and is referred to herein as the “Gax gene”. Theexpression of the Gax gene is restricted to the cardiovascular system,and in particular, to vascular smooth muscle cells where it functions asa negative regulator of cell proliferation.

[0031] Isolation of the Rat Gax cDNA

[0032] An adult rat aorta cDNA library in λ ZAP, from Stratagene, wasscreened with a 64-fold degenerate 29-mer oligonucleotide containingthree inosine residues directed at the most highly conserved region ofthe antennapedia homeodomain (helix 3), with the following sequence,where I represents inosine:5′-AA(A/G)ATITGGTT(T/C)CA(A/G)AA(C/T)(A/C)GI(A/C) GIATGAA-3′.

[0033] Recombinant phage colonies in Escherichia coli were adsorbed induplicate to nitrocellulose membranes and hybridized at 42° C. with thisoligonucleotide end labeled with (γ-32P)ATP in a mixture containing 0.5M sodium phosphate at pH 7.0, 7% sodium dodecyl sulfate, 1 mM EDTA, and1% bovine serum albumin. The filters were washed with a final stringencyof 0.5×SSC (1×SSC in 150 mM NaCl with 15 mM sodium citrate at pH7.0)-0.1% sodium dodecyl sulfate at 42° C. and exposed to X-ray film.Thirteen positive signals were isolated and rescreened until the cloneswere plaque purified. The plasmids containing the clones in λ ZAP vectorwere then excised by the protocol recommended by the manufacturer andsequenced on both strands with sequenase version 2.0 from United StatesBiochemicals. From 500,000 plaques, 13 positive clones were isolated, 12of which contained homeodomains. Nine of the isolated clones werederived from previously described homeobox genes: Hox-1.3, Hox-1.4,Hox-1.11, and rat homeobox R1b. However, three clones represented thecDNA designated herein as the “Gax” gene. Homology searches wereperformed via the GenBank and EMBL data bases, version 73, by using theBLAST algorithm (4).

[0034] Nucleotide Sequence of the Rat Gax Gene

[0035] The nucleotide sequence of the rat Gax gene is shown in FIG. 1.The cDNA encoding Gax is 2,244 base pairs in length, which correspondsto the size of the Gax transcript, that is the Gax mRNA, which is about2.3 to 2.4 kb as determined by Northern blot analysis. The Gax cDNA hasan open reading frame from nucleotide residues 197 to 1108 beginningwith an in-frame methionine that conforms to the eukaryotic consensussequence for the start of translation and is preceded by multiple stopcodons in all three reading frames. The open reading frame of the cDNApredicts a 33.6-kDa protein containing 303 amino acids with ahomeodomain from amino acid residues 185 to 245, as shown in FIG. 1. Toconfirm that this cDNA was capable of producing a protein product, theGax open reading frame was fused in frame to the pQE-9 E. coliexpression vector, from Qiagen, Inc., Chatsworth, Calif. and expressedin bacteria according to Hochuli, E., et. al. (1988) “Genetic Approachto Facilitate Purification of Recombinant Proteins with a Novel MetalChelate Adsorbent” Bio/Technology Vol., 6, pp. 1321-1325. E. colicontaining this plasmid expressed a new phosphorylated protein of about30 to about 36 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and extracts from these E.coli cells displayed a weak binding activity for the adenine and thyminerich, MHox-binding site in the creatine kinase M enhancer.

[0036] The cDNA encoding the rat Gax gene also contains a long3′-untranslated region, from bases 1109 to 2244, with a polyadenylationsignal at base 2237, as shown in FIG. 1. The region between amino acids87 and 184 contains 23 serine amino acids out of 88 amino acids and 10proline amino acids out of 88 amino acids and contains several consensussequences for phosphorylation by protein kinases. Gax also possesses astructural feature which is also found in several transcription factors,including homeodomain proteins, known as the CAX or Opa transcribedrepeat. The Opa transcribed repeat encodes a stretch of glutamines andhistidines; in the rat Gax gene it encodes 18 residues, of which 12 areconsecutive histidines. This motif is shared by other transcriptionfactors, such as the zinc finger gene YY-1, as well as by severalhomeobox genes, including H2.0, HB24, ERA-1 (Hox-1.6), Dual bar, andTes-1. The Gax protein may require post-translational modifications forfull activity, modifications that bacterially produced proteins do notundergo. Since the Gax protein has multiple consensus sites forphosphorylation by protein kinases, it is possible that its activity isactivated or otherwise modulated by phosphorylation at one or more ofthose sites.

[0037] The Gax Gene Maps to a Chromosome 12 of the Mouse Genome

[0038] Gax is located on chromosome 12 as shown in FIG. 2 of the mouseand is not a part of the Hox-1, Hox-2, Hox-3, or Hox-4 gene clusters,which are located on chromosomes 6, 11, 15, and 2, respectively,McGinnis, W., and R. Krumlauf, (1992) “Homeobox genes and AxialPatterning” Cell, Vol. 68, pp. 283-302. Also Gax does not cosegregatewith any other homeobox genes previously mapped in the interspecificbackcross. A comparison was done of the interspecific map of chromosome12 with a composite mouse linkage map that reports the map location ofmany uncloned mouse mutations using GBASE, a computerized data basemaintained at The Jackson. Laboratory, Bar Harbor, Me. The Gax genemapped in a region of the composite map that lacks mouse mutations witha phenotype that might be expected for an alteration in this locus.

[0039] The mouse chromosomal location of the Gax was determined byinterspecific backcross analysis using progeny generated by mating(C57BL/6J×Mus spretus) F₁ females and C57BL/6J males. The C57BL/6J andM. spretus DNAs were digested with several enzymes and analyzed bySouthern blot hybridization for informative restriction fragment lengthpolymorphisms with a rat cDNA Gax probe. The probe, a 1,155-bp rat cDNAclone, was labeled with (α-³²P)dCTP by using a random prime labeling kitfrom Amersham and washing was done with a final stringency of 0.2×SSCP(34)-0.1% sodium dodecyl sulfate, 65° C. A major fragment of 4.2 kb wasdetected in HincII-digested C57BL/6J DNA, and major fragments of 3.6 and2.7 kb were detected in HincII-digested M. spretus DNA. The 3.6-kb and2.7-kb M. spretus HincII restriction fragment length polymorphisms wereused to monitor the segregation of the Gax locus in backcross mice.Recombination distances were calculated by using the computer programSPRETUS MADNESS. Gene order was determined by minimizing the number ofrecombination events required to explain the allele distributionpatterns.

[0040] The mapping results indicated that the mouse Gax gene is locatedin the proximal region of mouse chromosome 12 linked to neuroblastomamyc-related oncogene 1 (Nmyc-1), the laminin B1 subunit gene (Lamb-1), aDNA segment, chromosome 12, the Nyu 1 gene (D12Nyu1), and the β-spectringene (Spnb-1). The ratios of the total number of mice exhibitingrecombinant chromosomes to the total number of mice analyzed for eachpair of loci and the most likely gene order are as follows:centromere-Nmyc-1-19/193-Lamb-1-9/166-Gax-10/166-D12Nyu1-19/185-Spnb-1.The recombination frequencies, expressed as genetic distances incentimorgans ± the standard error, are as follows:Nmyc-1-9.8±2.2-Lamb-1-5.4±1.8-Gax-6.0±1.9-D12Nyu1-10.3±2.2-Spnb-1.

[0041] Gax Gene Expression in Rat Tissue

[0042] It has been discovered that the Gax transcript is largelyconfined to the cardiovascular system, including the descending thoracicaorta, where it is expressed at higher levels than in other tissues, andthe heart. Gax gene expression was also detected in the adult lung andkidney where it is found in mesangial cells. No Gax gene expression wasdetected in the brain, liver, skeletal muscle, spleen, stomach, ortestes, nor was expression detected in the intestine or pancreas. Incontrast, the Gax gene was more widely expressed in the developingembryo, with the transcript detectable in the developing cardiovascularsystem, multiple mesodermal tissues, and some ectodermal tissues.

[0043] The 2.3-kb to 2.4-kb Gax RNA transcript was detected in smoothmuscle cells cultured from adult rat aorta, consistent with the in situhybridization findings and the fact that Gax was originally isolatedfrom a vascular smooth muscle library. The Gax transcript was alsodetected in rat vascular smooth muscle cells transformed by simian virus40. However, no Gax gene expression was detected in either of two celllines derived from embryonic rat aortic smooth muscle, A7r5 and A10. TheGax transcript was also not detected in NIH 3T3 fibroblasts, or humanforeskin fibroblasts. The Gax transcript was not detected in theskeletal muscle cell line C2C12. A relatively high level of Gax geneexpression was detected in cultured rat mesangial cells. Mesangial cellsshare many similarities to vascular smooth muscle cells, bothstructurally and functionally, and proliferate abnormally in renaldiseases such as glomerulonephritis and glomerulosclerosis.

[0044] Isolation of the Human Gax cDNA

[0045] The nucleotide sequence of the human Gax gene coding sequence isshown in FIG. 3. Approximately 1×10⁶ plaques from a human genomiclibrary in λFixII available from Stratagene were screened byconventional methods with a random primed EcoRI/BstXI fragmentencompassing nucleotides 485-1151 of the rat Gax cDNA. Two clonescontained the second exon of human Gax gene, having I82 base pairs.Using this coding information, the rest of the coding region was clonedby polymerase chain reaction methods.

[0046] Reverse transcriptase and polymerase chain reaction techniqueswere used to clone the 3′ end of the human cDNA. The template was wholehuman RNA isolated from human internal mammary artery isolated by TRIreagent from Molecular Research Center, Inc. The following reagentconcentrations were used in the reverse transcriptase reaction: 1 μg oftotal internal mammary artery RNA; 50 mM Tris-HCl pH 8.5; 30 mM KCl; 8mM MgCl₂; 1 mM DTT; 20 units RNAsin from Boehringer Mannheim; 1 mM eachof dATP, dTTP, dGTP, and dCTP; 0.5 μg random hexamers from BoehringerMannheim; and 40 u of AMV reverse transcriptase from BoehringerMannheim, in a total volume of 20 μL. This was incubated for 1 hour at42° C., heat inactivated, and then stored at −80° C. before use. Aninitial amplification of 10% of the reverse transcriptase reaction wasperformed with just the sense oligonucleotide primer, known as “H2” andAmpliwax™ PCR Gem 100 beads Perkin Elmer in a “hot start” procedureaccording to the directions of the manufacturer. The following reagentconcentrations were used: 50 mM KCL; 10 mM Tris-HCl at pH 8.3; 1.5 mMMgCl₂; 1 mg/mL gelatin; 0.2 mM each of dATP, dTTP, dGTP, and dCTP; 0.1μM primer(s); and 2.5 units of Taq polymerase from Boehringer Mannheimor Perkin Elmer in a volume of 100 μL (these conditions were usedthereafter unless noted). The cycling protocol was as follows: 94° C.for two minutes, then 30 cycles of 94° C. for 30 seconds, 45° C. for 1minute, and 72° C. for 1 minute. A second amplification was thenperformed on 10% of the primary reaction products using the H2 primerand a degenerate antisense oligonucleotide primer known as “P2B” againstthe carboxy terminal peptide. The cycling parameters were: 94° C. fortwo minutes followed by 30 cycles of 94° C. for 30 seconds, 40° C. for30 seconds, 50° C. for 1 minute and 72° C. for 1 minute. A product wasobserved of the correct size and following purification by Glass Fogfrom Bio101, on 2% Biogel agarose from Bio101 was blunt sub-cloned intoEcoRV digested BluescriptII SK+ vectors from Stratagene and sequenced tohigh resolution by Sequenase 2.0 from universal primers from UnitedStates Biochemical. Five individual clones were sequenced to eliminateany spurious Taq polymerase errors.

[0047] The 5′ end of the human coding region was amplified using ananchored polymerase chain reaction kit, available under the tradename“5′-Amplifinder RACE” from Clonetech according to the manufacturer'sinstructions. This method uses single stranded RNA ligase to ligate ananchor oligonucleotide onto the 3′ end of appropriately primed firststrand cDNA. Templates used were either human heart polyA+ RNA obtainedfrom Clonetech or polyA+ RNA isolated from primary cultures of humanvascular smooth muscle cells obtained from Clonetics. The polyA+ RNAfrom cultured vascular smooth muscle cells was purified with RNAzol Bfrom Biotecx using batch chromatography on oligo-dT latex beads fromQiagen. Both templates yielded amplified cDNAs and specific subcloneswere chosen solely by size. First strand RNA templates were prepared byeither specific priming or priming with random hexamers from BoehringerMannheim. In general, the specific primed templates yielded longerclones but could not be used for multiple step wise amplification of therest of the coding region.

[0048] Amplification from anchored templates using the sense anchorprimer and appropriate antisense specific primers was accomplished usingampliwax beads from Perkin Elmer and “hot start” polymerase chainreaction using the same reaction conditions as above, but with 0.2 μMprimers in a total volume of 50 μL. The cycling protocol was as follows:94° C. for 2 minutes then 30 cycles of 94° C. 45 seconds, 60° C. 45second, and 72° C. for 1.5 minutes, followed by a final extension of 72°C. for 10 minutes. Following a primary amplification, aliquots (10-20%)of the reactions were run out on 2% Biogel agarose from Bio101 and sizeselected. After purification by glass fog from Bio101, 1-10% of theelutes were reamplified (2°), usually with a nested primer. Productswere observed at this point and purified by glass fog as before andsequenced directly using a thermal cycling kit from New England Biolabs.Once the products were confirmed they were sub-cloned as describedabove. Between 5 to 8 individual clones from each of three sequentialamplifications were sequenced to eliminate spurious Taq polymeraseerrors and appropriate clones chosen for the finished molecule. Asummary of the primer pairs sense/antisense used to amplify the completecoding region follows: position Clone # Source of Template 1° 2° 5′-3′ 6 dN6 primed IMA whole RNA H2 H2/P2B 699-941  23 H2R primed HeartpolyA + RNA AP/H2R AP/H3 231-698 117 dN6 primed VSMC poly A + AP/H6AP/H6 119-230 RNA 131 dN6 primed VSMC poly A + AP/H6 AP/H7 1-118 RNA

[0049] Clones were pieced together 3′-5′ as follows: fragments 6 and 23share engineered. BglII sites; fragments 23 and 117 share a native SfaNIsite; fragment 117 has a native NcoI site which is compatible with anengineered BspHI site in fragment 131. Both engineered sites have asingle base change in the wobble base of leucine codons, as noted on thefinal sequence as shown in FIG. 3. Once assembled the molecule wasexcised by digestion with EcoRI and HindIII. The map in FIG. 4 shows themolecule and its orientation. TABLE 1 Primers Used to Amplify Human Gaxgene Primer Sequence 5′-3′ P2B TCA,IA(G/A),(G/A)TG,IGC,(G/A)TG,(T/C)TCH2 GCGCGC(AGATCT)CAC,TGA,AAG,ACA,GGT,AAA H2R TT,TAC,CTG,TCT,TTC,AGT,GAGH3 GCGCGC(AGATCT)AG,ATT,CAC,TGC,TAT,CTC,GTA H6GCGCGTGCCCCCTCTGATG,CTG,GCT,GGC,AAA,CAT,GT H7GCGCGC(TCTTGA)AGG,GCG,AGA,GAG,GAT,TGG,GA APCTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGATAG AnchorGGAGACTTCCAAGGTCTTAGCTATCA(CTTAAG)CAC

[0050] The Gax Gene Maps to a Novel Locus on Chromosome 7 in the HumanGenome

[0051] To determine the map location of Gax in the human genome, a 16.5kilobase pair fragment of the human genomic Gax gene in λFix II fromStratagene was purified with a Qiagen purification column according tothe directions of the manufacturer, and it was labeled with biotin11-dUTP by nick translation. Metaphase spreads of normal humanlymphocytes were prepared according to the methods of Fan, Y., Proc.Natl. Acad. Sci. (USA) Vol. 87, pp. 6223-6227 (1990). Fluorescence insitu hybridization and immunofluorescence detection were performedaccording to the methods of Pinkel, D., et. al., Proc. Natl. Acad. Sci.(USA) Vol. 83, pp. 2934-2938 (1986) and Testa, J. R., et al. Cytogenet.Cell. Genet. Vol. 60, pp. 247-249 (1992). Chromosome preparations werestained with diamidino-2-phenylindole and propidium iodide according toFan, Y. S., et. al., Proc. Natl. Acad. Sci. (USA) Vol. 87, pp.6223-6227(1990).

[0052] Forty metaphase spreads were examined with a Zeiss Axiophotfluorescence microscope, and fluorescent signals were detected on theshort arm of chromosome 7 in 34 of these spreads. All signals werelocated at p15-->p22, with approximately 70% of the signals at 7p21.Based on these data, Gax is the only homeoprotein known to map to thislocus.

[0053] Gax Gene Expression is Down-Regulated in Cultured Vascular SmoothMuscle Cells Upon Mitogen Stimulation

[0054] It has been found that the Gax gene is expressed in quiescentvascular smooth muscle cells. Since platelet derived growth factorhereinafter also referred to as “PDGF” and other growth factors regulatevascular smooth muscle proliferation and differentiation, differences inGax gene expression in response to PDGF and other mitogens such as fetalcalf serum were examined in cultured vascular myocytes.

[0055] Cultures of rat smooth muscle cells were obtained from the mediaof aortas isolated from adult male Sprague-Dawley rats. Cells wereseeded onto dishes in medium containing a 1:1 mixture of Dulbecco'smodified Eagle's medium and Ham's F12 and supplemented with 10% newborncalf serum. Once established, the cells were maintained at 37° C. in ahumidified atmosphere of 5% carbon dioxide, and subcultured within threedays after reaching confluence. Vascular smooth muscle cells werelabeled with monoclonal antibodies to smooth muscle a-actin from SigmaChemical Co. to verify identity.

[0056] The cultured cells were exposed to various mitogens asdiscussed-below. The cells were then harvested and the total mRNA wasextracted. The total RNA from rat cultured cells was prepared by theguanidine thiocyanate method according to Chomcynzski, P., and N.Sacchi, (1987) “Single-step Method of RNA Isolation by Acid GuanidiniumThiocyanate-phenol-chloroform Extraction” Anal. Biochem. Vol. 162, pp.156-159, fractionated on 1.2% agarose gels containing formaldehyde, andblotted onto nylon membranes. The RNA from cultured cells was separatedon 30-cm gels for transcript size determination and on 10-cm gels forother studies. Hybridizations were carried out at 65° C. in buffercontaining 0.5 M sodium phosphate at pH 7.0, 7% sodium dodecyl sulfate,1 mM EDTA, and 1% bovine serum albumin, using a cDNA probe labeled byrandom priming consisting of a truncated Gax cDNA lacking the 5′ end andthe CAX repeat, where the X may be cytosine or guanine. Probes forHox-1.3 and Hox-1.4 consisted of the cDNAs isolated from the rat aortalibrary, and the probe for Hox-1.11 consisted of the DraI-EcoRI fragmentof its cDNA. The blots were washed with a final stringency of 0.1 to0.2×SSC-0.1% sodium dodecylsulfate at 65° C. After the probings with thehomeobox probes were complete, the blots were rehybridized with a probeto rat glyceraldehyde 3-phosphate dehydrogenase hereinafter alsoreferred to as “GAPDH,” to demonstrate message integrity. Gax mRNA andGAPDH mRNA were quantified with a Molecular. Dynamics model 400SPhosphorImager to integrate bank intensities, or by scanningdensitometry of autoradiograms. In all quantitative comparisons of GaxmRNA levels between experimental groups, Gax mRNA levels were normalizedto the corresponding GAPDH level determined on the same blot, to accountfor differences in RNA loading.

[0057] Time Course of GAX Down-Regulation in Cultured Vascular SmoothMuscle Cells

[0058] Rat vascular smooth muscle cells, grown to a greater than about90% confluence, were placed in low-serum medium containing 0.5% calfserum for 3 days, to induce quiescence. At this time, the medium wasremoved from the cells and replaced with fresh medium containing either10% fetal calf serum or 10 ng/ml platelet derived growth factor fromhuman platelets. The cells were then incubated for the various times inthe presence of either the fetal calf serum or the PDGF. As a control,quiescent cells were incubated with fresh serum-free medium alone. Thecells exposed to PDGF were harvested at 0.25, 0.5, 1, 2, and 4 hours,and the cellular RNA isolated. The cells exposed to human fetal calfserum were harvested at 4, 24, and 48 hours. The Gax and the Hox mRNAlevels were determined by Northern blot analysis. Typical results areshown in FIGS. 5A and 5B.

[0059] A rapid down-regulation, that is a reduction in the amount of GaxmRNA, occurred in the vascular smooth muscle cells when they werestimulated with either fetal calf serum or PDGF as shown in FIGS. 5A and5B. The down-regulation ranged from 5- to nearly 20-fold, depending onthe mitogen used and the experiment. The down-regulation typicallyoccurred within 2 hours after stimulation with fetal calf serum or PDGF,and was maximal at 4 hours. Gax mRNA transcript levels began to recoversignificantly by approximately 24 hours and approached baseline between24 and 48 hours after stimulation. The rate of recovery varied with themagnitude of the initial down-regulation and the individual cell culturepreparations. While PDGF isolated from human platelets caused a rapiddown-regulation of Gax, it had little or no effect on Hox-1.3 mRNAlevels. Neither fetal calf serum nor any of the three isoforms of PDGFshowed any effect on the transcript levels of Hox-1.3, Hox-1.4, orHox-1.11, homeobox genes which were also isolated from the vascularsmooth muscle library.

[0060] Magnitude of Gax Down-Regulation Correlates with Potency ofMitogen

[0061] PDGF is a homodimer or heterodimer made of various combination oftwo chains, A and B. Thus, there are three isoforms of PDGF; PDGF-AA;PDGF-AB; and PDGF-BB; and they have differing potencies for stimulatingDNA synthesis in rat vascular smooth muscle cells. The PDGF-AA, PDGF-ABand PDGF-BB were compared for their effect on Gax expression. Quiescentrat vascular smooth muscle cell received 10 ng/ml of either PDGF-AA,PDGF-AB, PDGF-BB, or 10% fetal calf serum. After 0, 1, 2, 4 and 8 hoursthe cells were harvested and the Gax mRNA level determined. The resultsare shown in FIG. 6.

[0062] As shown in FIG. 6, PDGF-AA did not down-regulate Gax geneexpression in vascular smooth muscle cells, whereas the PDGF-AB andPDGF-BB isoforms, and the fetal calf serum reduced Gax gene expressionapproximately 10-fold by 4 hours. The greatest down-regulation occurredwith the fetal calf serum followed by that with PDGF-BB and PDGF-AB.

[0063] To determine whether the extent of Gax gene down-regulationcorrelated with the potency of the mitogen used to stimulate thevascular smooth muscle cells, the ability of the three PDGF isoforms andfetal calf serum to stimulate DNA synthesis was measured by ³H-thymidineuptake. Quiescent vascular smooth muscle cells were stimulated with oneof the three PDGF isoforms, at 10 mg/ml, or 10% fetal calf serum. Then 5μCi/ml ³H-thymidine was added to the cultures for 1 hour at various timepoints as shown in FIG. 7. The cells were harvested and the ³H-thymidineuptake was measured. The results are shown in FIG. 7.

[0064] The PDGF-AA at 10 ng/ml, which was ineffective in causing Gaxgene down-regulation, only weakly stimulated DNA synthesis as shown inFIG. 7. PDGF-AB and PDGF-BB both stimulated cell proliferation asmeasured by ³H-thymidine uptake at 15 hours. However, the fetal calfserum which was most effective at down regulating Gax gene expression,was also the most effective mitogen, that is it demonstrated thegreatest ³H-thymidine uptake.

[0065] Down-Regulation of the Gax Gene is Dependent on the Dose of theMitogen

[0066] Dose-response experiments were conducted by stimulating quiescentvascular smooth-muscle cells with either PDGF-AB, PDGF-BB or fetal calfserum at varying doses as shown in FIGS. 8 and 9. The effects on GaxmRNA levels were measured at 4 hours after mitogen stimulation. Theresults are shown in FIGS. 8 and 9.

[0067] As shown in FIG. 8, the dose response curves reveal that the 50%effective dose for Gax gene down-regulation 4 hours after PDGF-ABstimulation is between 4 and 8 ng/ml. The 50% effective dose for Gaxgene down regulation 4 hours after PDGF-BB stimulation is between 2 and5 ng/ml. The 50% effective dose for Gax down regulation 4 hours afterfetal calf serum is approximately 1%, as shown in FIG. 9. Furthermore,10% fetal calf serum suppresses Gax mRNA levels nearly 20-fold at 4hours, an effect larger than that of a maximal stimulatory dose ofPDGF-BB (30 ng/ml), which has a 10-fold effect, or of PDGF-AB, which hasa less than 8-fold effect, as shown in FIG. 6. Thus, the down-regulationof Gax gene induced by either fetal calf serum or the different isoformsof PDGF correlates well with their abilities to stimulate DNA synthesisas measured by ³H-thymidine uptake.

[0068] The Gax gene down-regulation is sensitive to low levels ofmitogen stimulation, which cause a significant decrease in Gax mRNAlevels. As shown in FIG. 9, stimulation of quiescent rat vascular smoothmuscle cells with 1% fetal calf serum caused a 40% decrease in Gax mRNAlevels after 4 hours. However, such stimulation increased ³H-thymidineuptake less than two-fold over that observed in quiescent vascularsmooth muscle cells (data not shown). Treatment with PDGF-BB at doses aslow as 2 ng/ml, also caused a detectable decrease in the Gax mRNA level.

[0069] Gax Expression is Up-Regulated or Induced when SynchronouslyGrowing Cells are Deprived of Serum

[0070] Sparsely plated vascular smooth muscle cells were grown in amedium containing 20% fetal calf serum, and then placed into serum freemedium. The RNA was harvested at various times from 0 to 25 hours. Thetotal mRNA was extracted and subjected to Northern Blot Analysis, thenthe mRNA transcript of Gax was quantified.

[0071] As shown in FIG. 10, the expression of the Gax gene was inducedfivefold in vascular smooth muscle cells within 24 hours after therapidly growing cells were placed in the serum-free medium. Thus,expression of Gax gene is regulated by the growth state of the cell, andits down-regulation is a prominent feature of the G₀/G₁ transition inthese cells.

[0072] Gax Protein Inhibits Mitogen-Induced S Phase Entry in VascularSmooth Muscle Cells

[0073] Production of Recombinant Proteins

[0074] To determine whether Gax gene exerts a negative control on cellgrowth in vascular smooth muscle cells, Gax gene was expressed as aglutathione S-transferase (hereinafter also referred to as “GST”) fusionprotein in bacteria and microinjected it into quiescent vascular smoothmuscle cells. To determine the effect of the Gax protein onserum-induced cell proliferation, the effect of GST-Gax protein wascompared to the effect of known protein regulators of cell growth.

[0075] To produce the Gax protein evaluated herein, the cDNA codingregions for Gax was fused in frame to the pGEX-2T expression vectorobtained from Pharmacia Biotechnology, and then expressed in E. coli.Specifically, GST-Gax was produced according to the following procedure:the coding region of Gax cDNA spanning from nucleotides 200-1108 wasamplified by polymerase chain reaction methods using the followingprimers: 5′GCGCGCGTCGACGAACACCCCCTCTTTGGC 3′ and5′GCGCGCAAGCTTTCATAAGTGTGCGTGCTC 3′

[0076] The resulting DNA was digested with SalI and HindIII restrictionenzymes and cloned into SalI and HindIII sites in the polylinker ofpGEM3-1T in vitro transcription translation vector described in Patel R.C. and Sen G. C. (1992) “Identification of the Double-strandedRNA-binding Domain in the Human Interferon-inducible Protein Kinase,” J.Biol. Chem. Vol. 267; pp. 7671-7676. The BamHI to NaeI fragment ofpGEM3-1T containing the Gax coding region was then sub-cloned into thesame sites of pGEX-2T. The pGEX-2T vector with the YY1 cDNA, used toproduce GST-YY1, was from Thomas Shenk at Princeton University.

[0077] The resultant glutathione S-transferase fusion proteins werepurified by affinity chromatography on glutathione-agarose beads. E.coli XL1-blue cells were then transformed with the appropriate plasmidand were grown to a density of 0.6-0.8 A₆₀₀ and induced with 0.5 mMisopropyl-B-D-thiogalectopyrenoside for 2 hours. The cells wereharvested and lysed by ultrasonic vibration in phosphate buffered salinecontaining 1% triton x-100, 1 mm PMSF and 5 μg/ml aprotinin. The lysatewas centrifuged at 15,000×g and the supernatant was collected. Thesupernatant was bound to the glutathione sepharose from Pharmacia (0.5ml of resin per 100 ml of bacterial culture) for 2 hours on a rotator at25 rpm. The slurry was pelleted by centrifugation at 1000×g for 2minutes, then washed twice with complete lysis buffer then washed twicewith lysis buffer lacking triton x-100. The bound protein was eluatedfor 30 minutes with phosphate buffered saline containing 10 mM reducedglutathione, from Sigma Chemical Company, 40 mM DTT and 150 mM NaCl.Purity of the GST-Gax protein was greater than 90% as determined bySDS-PAGE gels stained with Coomassie blue.

[0078] To produce recombinant MHox, its cDNA was fused in frame to thepQE-9 E. coli expression vector obtained from Qiagen, Inc., Chatsworth,Calif., then expressed in bacteria, and purified by adsorption to anickel column.

[0079] For microinjection, proteins were concentrated in a buffercontaining of 20 mM Tris, 40 mM KCl, 0.1 mM EDTA, 1 mMβ-mercaptoethanol, and 2% glycerol using Centricon-30 from Amiconmicroconcentrators. Concentrated proteins were stored in this buffer inaliquots at −80° C.

[0080] Microinjection and Cell Culture Methods

[0081] Microinjections were performed using a semiautomaticmicroinjection system from Eppendorf Inc. in conjunction with a NikonDiaphot phase contrast microscope. According to Peperkole, R., et al.(1988) Proc. Natl. Acad. Sci. USA Vol. 85, pp. 6758-6752, The injectionpressure was set at 70-200 hPa and the injection time was 0.3 to 0.6seconds.

[0082] After injection, cells were stimulated 24 hours with mediumcontaining 10% fetal calf serum, and the incorporation of5′-bromo-2′-deoxyuridine, hereinafter also referred to as “BrdU” wasmeasured with a cell proliferation kit according to the directions ofits manufacturer, Amersham. When fetal calf serum-stimulated BrdUlabeling was determined, BrdU was included for 24 hours with the mediumused to stimulate the cells. Where the ability of microinjected proteinsto stimulate growth in serum-poor medium was measured, cells wereincubated 24 hours in the same low serum medium used to inducequiescence, but supplemented with BrdU. After labeling, the cells werefixed with acid-ethanol, and the percentage of nuclei positive for BrdUuptake was determined for protein-injected and buffer-injected cells.The percent of cell growth inhibition was calculated according to thefollowing formula:${\% \quad {Inhibition}} = {\frac{\frac{C\quad L}{C\quad T} - \frac{I\quad L}{I\quad T}}{\frac{C\quad L}{C\quad T}} \times 100}$

[0083] where IL represents the number of injected labeling positive forBrdU; IT, the total number of injected cells; CL the number ofcontrol-injected cells labeling with BrdU; CT, the total number ofcontrol-injected cells counted. With this equation, inhibition ofmitogen-induced entry into S phase is represented by a positive numberand stimulation of cell growth is represented by a negative number.

[0084] Evaluation of Gax Protein

[0085] To determine if the Gax protein inhibits the entry of mitogenstimulated vascular smooth muscle cells into S-phase, the effect of theGax protein was compared to proteins known to effect cell proliferation,and to control proteins. Such comparison proteins include a neutralizingantibody against ras, “Y13-259,” which is highly effective in blocking Sphase entry when microinjected into NIH3T3 cells; the transcriptionfactor MHox, a homeodomain protein unlikely to have an inhibitory effecton cell proliferation; and YY1, a zinc finger transcription factorunlikely to have a negative effect on cell growth.

[0086] Quiescent rat vascular smooth muscle cells were microinjectedwith either. 0.6 mg/ml GST-Gax protein; 1.6 mg/ml MHox; 1.2 mg/ml YY1, 8mg/ml Y13-259; 2 mg/ml GST alone; or 8 mg/ml mouse anti-human IgG. Thecells were then stimulated for 24 hours with 10% fetal calf serum inmedium containing BrdU. After 24 hours, the fraction of nuclei labelingwith BrdU was determined and percentage inhibition of S-phase entrycalculated. The results are summarized in Table 2. TABLE 2 Effect ofMicroinjected Proteins on the Serum-induced Proliferation of VascularSmooth Muscle Cells Mean % Inhibition of Total Number FCS-stimulatedNumber of of Cells Growth ± Treatment Experiments Examined StandardError Antibody Y13-259 2 328 60.8 ± 3.9 Mouse anti-human 3 330 −3.4 ±4.5 IgG GST-Gax 15 2943 42.7 ± 3.3 MHox 2 236 −5.3 ± 9.3 GST-YY1 5 306 0.0 ± 12.2 GST 7 1144 −2.6 ± 2.1

[0087] As shown in Table 2, the GST-Gax protein inhibited vascularsmooth muscle cell entry into S-phase by 42.7%. The GST Gax proteineffect on mitogen-stimulated entry into S phase is specific. The otherinjected proteins GST, YY1, MHox and the mouse anti-human IgG failed toinhibit vascular smooth muscle cell growth. In comparison to the GST-Gaxprotein, the antibody Y13-259, as anticipated, significantly decreasedmitogen-induced cell proliferation. Vascular smooth muscle cellsmicroinjected with Y13259 demonstrated a 61±4% decrease in cell entryinto S-phase

[0088] Gax Protein Inhibits Vascular Smooth Muscle Cell Proliferation ina Dose-Dependent Manner.

[0089] To determine the concentration of microinjected GST-Gax requiredto inhibit vascular smooth muscle cell growth, solutions containingdifferent concentrations of GST-Gax protein were microinjected intoquiescent vascular smooth muscle cell and the effects onmitogen-stimulated entry into S phase examined. Specifically, vascularsmooth muscle cells were rendered quiescent by incubation in mediumcontaining 0.5% calf serum for three days. The cells were microinjectedwith varying concentrations of GST-Gax, and stimulated with 10% fetalcalf serum, and labeled with BrdU. After 24 hours, the percentageinhibition of cell proliferation was determined. Each data pointrepresents the mean±standard error of 3-5 experiments in which 100-200cells per experimental group were injected.

[0090] As shown in FIG. 11, the cellular growth inhibition by theGST-Gax protein is dose dependent. Little or no growth inhibition wasobserved when 0.2 mg/ml GST-Gax protein was injected. The maximal growthinhibition was obtained with approximately 0.5 mg/ml of the GST-Gaxprotein.

[0091] Gax Inhibits Proliferation of Several Cell Types

[0092] To determine whether the GST-Gax protein inhibits growth in othercells types, the GST-Gax protein was microinjected into quiescentSV40-transformed vascular smooth muscle cells, BALBc3T3 cells, NIH3T3cells, human vascular smooth muscle cells, and human fibroblasts. TheSV40 transformed cell line was derived from rat vascular smooth musclecells transformed with the SV40 large T antigen. These cells, whileimmortalized, retain many differentiated characteristics ofuntransformed vascular smooth muscle cells. The cells were microinjectedwith either 0.6 mg/ml GST-Gax protein or 2 mg/ml GST were thenstimulated with 10% fetal calf serum, and labeled for 24 hours withBrdU. The results are shown in Table 3. TABLE 3 EFFECT OF MICROINJECTEDGST-GAX PROTEIN ON CELL PROLIFERATION IN DIFFERENT CELL TYPES Mean &Range Inhibition Mitotic Number Number of of FCS- Index in GST-GAX ofCells Stimulated Response Cell type protein Experiments Examined Growthto FCS SV40- Yes 4 448 27.2 ± 2.0 — transformed VSMC SV40- No N/A 0.60 ±0.02 transformed VSMC BALB/c Yes 4 464  30.5 ± 10.9 — 3T3 cells BALB/cNo N/A 0.64 ± 0.03 3T3 cells NIH3T3 cells Yes 4 420 23.2 ± 1.8 — NIH3T3cells No N/A 0.70 ± 0.02 Human VSMC Yes 3 506 46.6 ± 8.1 — Human VSMC NoN/A 0.33 ± 0.02 Human Yes 3 336 44.5 ± 2.1 — fibroblasts Human No N/A0.36 ± 0.01 fibroblasts

[0093] SV40-transformed vascular smooth muscle cell proliferation wasinhibited by GST-Gax protein, as shown in Table 3. The GST-Gax proteinalso inhibited the proliferation of fibroblast cell lines NIH3T3 andBALB/c. 3T3. GST-Gax protein also inhibited the proliferation of humancells, specifically human vascular smooth muscle cells and humanforeskin fibroblasts. These results indicate that Gax action is not celltype-specific, although there are differences in the extent inhibitionamong the different cell types. The Among the human cells, the GST-Gaxprotein exhibits maximal inhibition in vascular smooth muscle cells, thecell type in which the Gax gene is normally expressed. Similarly amongthe rat cells, the GST-Gax protein exhibits maximal inhibition invascular smooth muscle cells, the cell type in which the Gax gene isnormally expressed.

[0094] An Oncogenic Ras Protein can Reverse Growth Inhibition Caused bythe Gax Protein

[0095] To characterize the mechanism of the growth inhibition conferredby the GST-Gax protein, the effects of GST-Gax protein and thetransforming oncoprotein, the ras mutant Ras(Leu-61) were compared bymicroinjecting these proteins into rat vascular smooth muscle cells. Asolution containing both 0.5 mg/ml GST-Gax protein and 0.5 mg/mlRas(Leu-61) was microinjected into quiescent vascular smooth musclecells. For comparison, other vascular smooth muscle cells receivedeither 0.5 mg/ml GST-Gax protein or 0.5 mg/ml Ras(Leu-61) or 0.5 mg/mlGST. The injected cells were then incubated for 24 hours with mediumcontaining 10% fetal calf serum and BrdU. The results are shown in FIG.12.

[0096] As shown in FIG. 12, when Ras(Leu-61) alone was injected, therewas an increase in BrdU-labeling as compared to both control-injectedcells. In cells injected with GST-Gax protein, growth was inhibited 39%.When the GST-Gax protein and Ras(Leu-61) were coinjected in the cells,the Ras(Leu-61) reversed the growth inhibitory effects of the GST-Gaxprotein, and the percentage of cells staining positive for BrdU in cellsreceiving both the Ras(Leu-61) and GST-Gax protein were nearly identicalto that observed in cells receiving just the Ras(Leu-61). Thus, the Rasoncoprotein completely reversed the effect of the GST-Gax proteinestablishing that the presence of GST-Gax protein is not toxic to cells.

[0097] The Gax Protein Inhibits Cell Growth when Microinjected Beforethe GI to S Boundary

[0098] To determine the point in the cell cycle when the Gax gene exertsits growth inhibitory effects, the time of S phase onset was determinedin rat vascular smooth muscle cells. The vascular smooth muscle cellswere stimulated with 10% fetal calf serum and pulse labeled with 10mCi/ml ³H-thymidine for one hour at different times after stimulation.Serarate cultures of the cells were microinjected with GST-Gax proteinat various times after receiving 10% fetal calf serum and labeled withBrdU between 10 and 24 hours after receiving the fetal calf serum.Percent inhibition of S-phase entry was determined at each time point.The results are shown in FIG. 13.

[0099] As shown in FIG. 13, S phase onset, indicated by the uptake of³H-thymidine, occured at approximately 16-18 hours after mitogenstimulation. GST-Gax protein significantly inhibited vascular smoothmuscle cell entry into the S phase when microinjected at any time fromstimulation up until approximately 12 hours. However, GST-Gax proteinwas ineffective when injected at 15 hours. Thus it appears that the Gaxgene inhibits a critical step in cell cycle progression prior to theG₁/S boundary; perhaps before the restriction point in G₁ whereeukaryotic cells are irreversibly committed to entering the S phase.

[0100] Gax Gene Expression is Rapidly Down Regulated in Vivo Upon AcuteBlood Vessel Injury

[0101] The Gax gene expression in normal blood vessels and in injuredblood vessels was compared to determine whether Gax gene down-regulationoccurs in response to injury-induced smooth muscle cell proliferation invivo. Adult male Sprague-Dawley rats were subject to acute vessel injuryby balloon de-endothelialization in the carotid arteries according tothe methods of Majesky, M. W., et al. J. Cell. Biol. (1990) Vol. 111,pp. 2149-2158. The expression levels of Gax, that is, the mRNA levels,were assessed relative to that of glyceraldehyde 3-phosphatedehydrogenase (hereinafter also referred to as “G3”) by a quantitativepolymerase chain reaction. At various times following balloonde-endothelialization the rats were sacrificed and the total RNA wasisolated from the vascular smooth muscle tissues using the TRI reagentfrom Molecular Research Center, Inc. The cDNA was synthesized from theextracted RNA with MMLV reverse transcriptase from Bethesda ResearchLabs. Aliquots of the cDNA pools were subjected to polymerase chainreaction amplification with AmpliTaq DNA polymerase from Perkin-Elmer inthe presence of α32P-dCTP with the following cycle conditions: 94° C.for 20 seconds, 55° C. for 20 seconds, and 72° C. for 20 seconds. Thefinal cycle had an elongation step at 72° C. for 5 minutes. The primersfor the rat Gax amplification were: 5′-CCCGCGCGGCTTTTACATTAGGAGT-3′ and5′-GCTGGCAAACATGCCCTCCTCATTG-3′. The primers for the rat G3 gene were5′-TGATGGCATGGACTGTGGTCATGA-3′ and 5′-TGATGGCATGGACTGTGGTCATGA-3′. TheGax cDNA was amplified for 30 cycles, and G3 was amplified for 25 cyclesin the same reaction vessels. The amount of a radioactive labelincorporated into the amplified cDNA and G3 fragments was determined bysubjecting the fragments to electrophoresis on a 1% agarose gel, thenexcising the bands and liquid scintillation counting. Since the mRNAlevels of glyceraldehyde 3-phosphate dehydrogenase remain relativelyconstant following this procedure (see J. M. Miano et al. 1990, Am. J.Path. 137, 761-765)., the ratio of radiolabel incorporation into theGax-derived amplified bands and the G3-derived amplified bands correctsfor differences arising from the efficiency of RNA extraction from thedifferent animals, and it provides a measure of Gax mRNA levels in thenormal and injured vascular tissues. These ratios are plotted in FIG.14.

[0102] As shown in FIG. 14, the Gax mRNA expression was down-regulatedin response to acute vessel injury by as much as a factor of 20. Thisdown-regulation was rapid and appeared complete by 2 hours, the firsttime-point following the de-endothelialization procedure. Collectively,these data corroborate the Gax gene down-regulation in cultures ofvascular smooth muscle cells following growth factor stimulation.Further, these data show that Gax gene expression is an early marker ofthe cell cycle activity associated with the initiation of vascularrestenosis, and they indicate that Gax has a regulatory role followingblood vessel injury.

[0103] The present invention includes: the DNA sequences encoding aprotein, or portion thereof, which inhibits vascular smooth muscle cellproliferation; the messenger RNA transcript of such DNA sequence; and anisolated protein which inhibits vascular smooth muscle cell growth.

[0104] For example, the DNA sequences include: DNA molecules which, butfor the degeneracy of the genetic code would hybridize to DNA encodingthe Gax protein, thus the degenerate DNA which encodes the Gax protein;DNA strands complementary to DNA sequences encoding the Gax protein orportions thereof including DNA in FIGS. 1 and 3 or portions thereof;heterologous DNA having substantial sequence homology to the DNAencoding the Gax protein, including the DNA sequences in FIGS. 1 and 3or portions thereof.

[0105] The isolated protein includes, for example, portions of the Gaxprotein; the Gax protein of animals other than rat and human; andproteins or portions thereof having substantially the same amino acidsequence as shown in FIGS. 1 and 3 or portions thereof.

1 19 2244 base pairs nucleic acid both linear cDNA NO NO CDS 197..1108 1GTCAAGTGTT TATACGTGCA GGAGACTGGC CGCTCGGCTC AGGACTGGGA TTAGCGGGCT 60CTGCTCAAAC CCGCGCGGCT TTTACATTAG GAGTGAGTGG GGGAGAGTCC TAGGATTTCT 120AGTGAAAAGT GACAGCGCTT GGTGGACTTT GGGACCTTCG TGAAGTCTTC TGCTTGGAAG 180CTGAGACTTG CATGCC ATG GAA CAC CCC CTC TTT GGC TGC CTG CGC AGC 229 MetGlu His Pro Leu Phe Gly Cys Leu Arg Ser 1 5 10 CCC CAC GCC ACA GCG CAAGGC TTG CAC CCC TTC TCG CAG TCT TCT CTG 277 Pro His Ala Thr Ala Gln GlyLeu His Pro Phe Ser Gln Ser Ser Leu 15 20 25 GCC CTC CAT GGA AGA TCT GACCAC ATG TCC TAC CCC GAA CTC TCC ACA 325 Ala Leu His Gly Arg Ser Asp HisMet Ser Tyr Pro Glu Leu Ser Thr 30 35 40 TCT TCC TCG TCT TGC ATA ATC GCGGGA TAC CCC AAT GAG GAG GGC ATG 373 Ser Ser Ser Ser Cys Ile Ile Ala GlyTyr Pro Asn Glu Glu Gly Met 45 50 55 TTT GCC AGC CAG CAT CAC AGG GGG CACCAC CAC CAC CAC CAC CAC CAC 421 Phe Ala Ser Gln His His Arg Gly His HisHis His His His His His 60 65 70 75 CAT CAC CAC CAC CAG CAG CAG CAG CACCAG GCT CTG CAA AGC AAC TGG 469 His His His His Gln Gln Gln Gln His GlnAla Leu Gln Ser Asn Trp 80 85 90 CAC CTC CCG CAG ATG TCC TCC CCG CCA AGCGCG GCC CGG CAC AGC CTT 517 His Leu Pro Gln Met Ser Ser Pro Pro Ser AlaAla Arg His Ser Leu 95 100 105 TGC CTG CAG CCT GAT TCC GGA GGG CCC CCGGAG CTG GGG AGC AGC CCT 565 Cys Leu Gln Pro Asp Ser Gly Gly Pro Pro GluLeu Gly Ser Ser Pro 110 115 120 CCG GTC CTG TGC TCC AAC TCT TCT AGC CTGGGC TCC AGC ACC CCG ACC 613 Pro Val Leu Cys Ser Asn Ser Ser Ser Leu GlySer Ser Thr Pro Thr 125 130 135 GGA GCC GCG TGC GCA CCA AGG GAT TAT GGCCGT CAA GCG CTG TCA CCC 661 Gly Ala Ala Cys Ala Pro Arg Asp Tyr Gly ArgGln Ala Leu Ser Pro 140 145 150 155 GCA GAA GTG GAG AAG AGA AGT GGC AGCAAA AGA AAA AGC GAC AGT TCA 709 Ala Glu Val Glu Lys Arg Ser Gly Ser LysArg Lys Ser Asp Ser Ser 160 165 170 GAT TCC CAG GAA GGA AAT TAC AAG TCAGAA GTG AAC AGC AAA CCT AGG 757 Asp Ser Gln Glu Gly Asn Tyr Lys Ser GluVal Asn Ser Lys Pro Arg 175 180 185 AGG GAA AGA ACA GCT TTC ACC AAA GAGCAA ATC AGA GAA CTT GAG GCA 805 Arg Glu Arg Thr Ala Phe Thr Lys Glu GlnIle Arg Glu Leu Glu Ala 190 195 200 GAG TTC GCC CAT CAT AAC TAT CTG ACCAGA CTG AGA AGA TAT GAG ATA 853 Glu Phe Ala His His Asn Tyr Leu Thr ArgLeu Arg Arg Tyr Glu Ile 205 210 215 GCG GTG AAC CTA GAC CTC ACT GAA AGACAG GTG AAA GTG TGG TTC CAG 901 Ala Val Asn Leu Asp Leu Thr Glu Arg GlnVal Lys Val Trp Phe Gln 220 225 230 235 AAC AGG AGA ATG AAG TGG AAG CGGGTC AAG GGG GGA CAA CAA GGA GCT 949 Asn Arg Arg Met Lys Trp Lys Arg ValLys Gly Gly Gln Gln Gly Ala 240 245 250 GCA GCC CGA GAA AAG GAA CTG GTGAAT GTG AAA AAG GGA ACA CTT CTT 997 Ala Ala Arg Glu Lys Glu Leu Val AsnVal Lys Lys Gly Thr Leu Leu 255 260 265 CCA TCA GAG CTG TCA GGA ATT GGTGCA GCC ACC CTC CAG CAG ACA GGG 1045 Pro Ser Glu Leu Ser Gly Ile Gly AlaAla Thr Leu Gln Gln Thr Gly 270 275 280 GAC TCA CTA GCA AAT GAC GAC AGTCGC GAT AGT GAC CAC AGC TCT GAG 1093 Asp Ser Leu Ala Asn Asp Asp Ser ArgAsp Ser Asp His Ser Ser Glu 285 290 295 CAC GCA CAC TTA TGATACATACAGAGACCAGC TCCGTTCTCA GGAAAGCACC 1145 His Ala His Leu 300 ATTGTGATGGCAAATCTCAC CCAAACATCG TTTACATGGC AGATGACTGT GGCAGTGTTG 1205 CTTAATATAATTAAACGCAG GCATCTCAAG TCTGTTTCTC ATGATTGATA GAAGGTTTAC 1265 ACTAAGTGCCTCTTATTGAA GATGCTTCCA CAGTGAAATT GGAGAAAGTG AACATATCTA 1325 AATATACTTGTTCCTTATAT GACAGAGAGG GAGATGAATG TTTGCTTTGG CTTGCACTGA 1385 AAATTAAATTGCTACCAAGA GCAAACTCGG TAAGACATTT TGACTCAAGT TGTCTCCAGA 1445 GTGAAGATGTTATAGAAATG CTTTGAACAT TCCAGTTGTA CCAGGTCATG TGTGTGACAC 1505 TGGGCAGGTATTTGCTTTTG CTTGCACTGA AACTTAAACT GCTATCAAGT TAACCCATGA 1565 AATAGTTTATCTTGAACAGC CACAGTGCCT GAAATCACCA AGTGGATATA AAATGAACTG 1625 AAATTCTGTATATATTACTC CTAAGTCATT TTCCTGTCTT CACTAATTTT AGCAAATGCA 1685 TTCATATTAGCTGATGAAAA TAGGCTTTCC CGTGGACAAA TGCAGCCAGC TTCTTGTATT 1745 TTTATACATTTTTTTGTCAG TCAGAGACAT CAGTATGTGC TTACTTGTGT TCAAGTAGAG 1805 GAAATGCAGTAGAGTCTGAT AGGACATATT CTTGGTACCA CAGACAAAAC AAATCTTCTG 1865 TTGCATTGACTATCAACTGC TGCAGATACA TTAGAGAACA CACCTAGCCC CCCTCCAGCC 1925 TCCCTCTGTTATCGCTCGAA GACATTAGCG TCATAGGCAA GTAGTTACCT TGCCAAATGA 1985 GTCTTGTGTGGCAGATGTCT GATTTTGTAT CTTTAAACTG TTAATGGTAT GTGTCTGCTT 2045 CAGTTAACAGGGAAAAAGAT TTCTTCCTCA TTGTTTATGA TACAAAACCC AAGTGCCAAA 2105 CAAAGCTAGTTCTTCAAGGG ATAGATGAGA AACTGAATGT CTGACAAGTA GACTCAGCGA 2165 AAATACATTATTTTTCAGAG GCTGTGTATT CATGCAGTAC AAGTCCTTGT ATTTTGTAAA 2225 AAAAAAAGTTAAATAAATG 2244 303 amino acids amino acid linear protein 2 Met Glu HisPro Leu Phe Gly Cys Leu Arg Ser Pro His Ala Thr Ala 1 5 10 15 Gln GlyLeu His Pro Phe Ser Gln Ser Ser Leu Ala Leu His Gly Arg 20 25 30 Ser AspHis Met Ser Tyr Pro Glu Leu Ser Thr Ser Ser Ser Ser Cys 35 40 45 Ile IleAla Gly Tyr Pro Asn Glu Glu Gly Met Phe Ala Ser Gln His 50 55 60 His ArgGly His His His His His His His His His His His His Gln 65 70 75 80 GlnGln Gln His Gln Ala Leu Gln Ser Asn Trp His Leu Pro Gln Met 85 90 95 SerSer Pro Pro Ser Ala Ala Arg His Ser Leu Cys Leu Gln Pro Asp 100 105 110Ser Gly Gly Pro Pro Glu Leu Gly Ser Ser Pro Pro Val Leu Cys Ser 115 120125 Asn Ser Ser Ser Leu Gly Ser Ser Thr Pro Thr Gly Ala Ala Cys Ala 130135 140 Pro Arg Asp Tyr Gly Arg Gln Ala Leu Ser Pro Ala Glu Val Glu Lys145 150 155 160 Arg Ser Gly Ser Lys Arg Lys Ser Asp Ser Ser Asp Ser GlnGlu Gly 165 170 175 Asn Tyr Lys Ser Glu Val Asn Ser Lys Pro Arg Arg GluArg Thr Ala 180 185 190 Phe Thr Lys Glu Gln Ile Arg Glu Leu Glu Ala GluPhe Ala His His 195 200 205 Asn Tyr Leu Thr Arg Leu Arg Arg Tyr Glu IleAla Val Asn Leu Asp 210 215 220 Leu Thr Glu Arg Gln Val Lys Val Trp PheGln Asn Arg Arg Met Lys 225 230 235 240 Trp Lys Arg Val Lys Gly Gly GlnGln Gly Ala Ala Ala Arg Glu Lys 245 250 255 Glu Leu Val Asn Val Lys LysGly Thr Leu Leu Pro Ser Glu Leu Ser 260 265 270 Gly Ile Gly Ala Ala ThrLeu Gln Gln Thr Gly Asp Ser Leu Ala Asn 275 280 285 Asp Asp Ser Arg AspSer Asp His Ser Ser Glu His Ala His Leu 290 295 300 941 base pairsnucleic acid both linear cDNA NO NO CDS 33..941 3 GTCTTCTACC TGGAACCCGAAACTTGCATG CT ATG GAA CAC CCG CTC TTT GGC 53 Met Glu His Pro Leu Phe Gly1 5 TGC CTG CGC AGC CCT CAC GCC ACG GCG CAA GGC TTG CAC CCG TTC TCC 101Cys Leu Arg Ser Pro His Ala Thr Ala Gln Gly Leu His Pro Phe Ser 10 15 20CAA TCC TCT CTC GCC CTC CAT GGA AGA TCT GAC CAT ATG TCT TAC CCC 149 GlnSer Ser Leu Ala Leu His Gly Arg Ser Asp His Met Ser Tyr Pro 25 30 35 GAGCTC TCT ACT TCT TCC TCA TCT TGC ATA ATC GCG GGA TAC CCC AAC 197 Glu LeuSer Thr Ser Ser Ser Ser Cys Ile Ile Ala Gly Tyr Pro Asn 40 45 50 55 GAAGAG GAC ATG TTT GCC AGC CAG CAT CAC AGG GGG CAC CAC CAC CAC 245 Glu GluAsp Met Phe Ala Ser Gln His His Arg Gly His His His His 60 65 70 CAC CACCAC CAT CAC CAC CAT CAG CAG CAG CAG CAC CAG GCT CTG CAA 293 His His HisHis His His His Gln Gln Gln Gln His Gln Ala Leu Gln 75 80 85 ACC AAC TGGCAC CTC CCG CAG ATG TCT TCC CCA CCG AGT GCG GCT CGG 341 Thr Asn Trp HisLeu Pro Gln Met Ser Ser Pro Pro Ser Ala Ala Arg 90 95 100 CAT AGC CTCTGC CTC CAG CCC GAC TCT GGA GGG CCC CCA GAG TTG GGG 389 His Ser Leu CysLeu Gln Pro Asp Ser Gly Gly Pro Pro Glu Leu Gly 105 110 115 AGC AGC CCGCCC GTC CTG TGC TCC AAC TCT TCC AGC TTG GGC TCC AGC 437 Ser Ser Pro ProVal Leu Cys Ser Asn Ser Ser Ser Leu Gly Ser Ser 120 125 130 135 ACC CCGACT GGG GCC GCG TGC GCG CCG GGG GAC TAC GGC CGC CAG GCA 485 Thr Pro ThrGly Ala Ala Cys Ala Pro Gly Asp Tyr Gly Arg Gln Ala 140 145 150 CTG TCACCT GCG GAG GCG GAG AAG CGA AGC GGC GGC AAG AGG AAA AGC 533 Leu Ser ProAla Glu Ala Glu Lys Arg Ser Gly Gly Lys Arg Lys Ser 155 160 165 GAC AGCTCA GAC TCC CAG GAA GGA AAT TAC AAG TCA GAA GTC AAC AGC 581 Asp Ser SerAsp Ser Gln Glu Gly Asn Tyr Lys Ser Glu Val Asn Ser 170 175 180 AAA CCCAGG AAA GAA AGG ACA GCA TTT ACC AAA GAG CAA ATC AGA GAA 629 Lys Pro ArgLys Glu Arg Thr Ala Phe Thr Lys Glu Gln Ile Arg Glu 185 190 195 CTT GAAGCA GAA TTT GCC CAT CAT AAT TAT CTC ACC AGA CTG AGG CGA 677 Leu Glu AlaGlu Phe Ala His His Asn Tyr Leu Thr Arg Leu Arg Arg 200 205 210 215 TACGAG ATA GCA GTG AAT CTG GAT CTC ACT GAA AGA CAG GTA AAA GTC 725 Tyr GluIle Ala Val Asn Leu Asp Leu Thr Glu Arg Gln Val Lys Val 220 225 230 TGGTTC CAA AAC AGG CGG ATG AAG TGG AAG AGG GTA AAG GGT GGA CAG 773 Trp PheGln Asn Arg Arg Met Lys Trp Lys Arg Val Lys Gly Gly Gln 235 240 245 CAAGGA GCT GCG GCT CGG GAA AAG GAA CTG GTG AAT GTG AAA AAG GGA 821 Gln GlyAla Ala Ala Arg Glu Lys Glu Leu Val Asn Val Lys Lys Gly 250 255 260 ACACTT CTC CCA TCA GAG CTG TCG GGA ATT GGT GCA GCC ACC CTC CAG 869 Thr LeuLeu Pro Ser Glu Leu Ser Gly Ile Gly Ala Ala Thr Leu Gln 265 270 275 CAAACA GGG GAC TCT ATA GCA AAT GAA GAC AGT CAC GAC AGT GAC CAC 917 Gln ThrGly Asp Ser Ile Ala Asn Glu Asp Ser His Asp Ser Asp His 280 285 290 295AGC TCA GAG CAC GCC CAC CTC TGA 941 Ser Ser Glu His Ala His Leu 300 302amino acids amino acid linear protein 4 Met Glu His Pro Leu Phe Gly CysLeu Arg Ser Pro His Ala Thr Ala 1 5 10 15 Gln Gly Leu His Pro Phe SerGln Ser Ser Leu Ala Leu His Gly Arg 20 25 30 Ser Asp His Met Ser Tyr ProGlu Leu Ser Thr Ser Ser Ser Ser Cys 35 40 45 Ile Ile Ala Gly Tyr Pro AsnGlu Glu Asp Met Phe Ala Ser Gln His 50 55 60 His Arg Gly His His His HisHis His His His His His His Gln Gln 65 70 75 80 Gln Gln His Gln Ala LeuGln Thr Asn Trp His Leu Pro Gln Met Ser 85 90 95 Ser Pro Pro Ser Ala AlaArg His Ser Leu Cys Leu Gln Pro Asp Ser 100 105 110 Gly Gly Pro Pro GluLeu Gly Ser Ser Pro Pro Val Leu Cys Ser Asn 115 120 125 Ser Ser Ser LeuGly Ser Ser Thr Pro Thr Gly Ala Ala Cys Ala Pro 130 135 140 Gly Asp TyrGly Arg Gln Ala Leu Ser Pro Ala Glu Ala Glu Lys Arg 145 150 155 160 SerGly Gly Lys Arg Lys Ser Asp Ser Ser Asp Ser Gln Glu Gly Asn 165 170 175Tyr Lys Ser Glu Val Asn Ser Lys Pro Arg Lys Glu Arg Thr Ala Phe 180 185190 Thr Lys Glu Gln Ile Arg Glu Leu Glu Ala Glu Phe Ala His His Asn 195200 205 Tyr Leu Thr Arg Leu Arg Arg Tyr Glu Ile Ala Val Asn Leu Asp Leu210 215 220 Thr Glu Arg Gln Val Lys Val Trp Phe Gln Asn Arg Arg Met LysTrp 225 230 235 240 Lys Arg Val Lys Gly Gly Gln Gln Gly Ala Ala Ala ArgGlu Lys Glu 245 250 255 Leu Val Asn Val Lys Lys Gly Thr Leu Leu Pro SerGlu Leu Ser Gly 260 265 270 Ile Gly Ala Ala Thr Leu Gln Gln Thr Gly AspSer Ile Ala Asn Glu 275 280 285 Asp Ser His Asp Ser Asp His Ser Ser GluHis Ala His Leu 290 295 300 29 base pairs nucleic acid single linearcDNA NO NO modified_base 6 /mod_base= i modified_base 21 /mod_base= imodified_base 24 /mod_base= i 5 AARATWTGGT TYCARAAYMG WMGWATGAA 29 18base pairs nucleic acid single linear cDNA NO YES modified_base 4/mod_base= i 6 TCAWARRTGW GCRTGYTC 18 30 base pairs nucleic acid singlelinear cDNA NO NO 7 GCGCGCAGAT CTCACTGAAA GACAGGTAAA 30 20 base pairsnucleic acid single linear cDNA NO YES 8 TTTACCTGTC TTTCAGTGAG 20 32base pairs nucleic acid single linear cDNA NO YES 9 GCGCGCAGATCTAGATTCAC TGCTATCTCG TA 32 36 base pairs nucleic acid single linearcDNA NO YES 10 GCGCGTGCCC CCTCTGATGC TGGCTGGCAA ACATGT 36 32 base pairsnucleic acid single linear cDNA NO 11 GCGCGCTCTT GAAGGGCGAG AGAGGATTGGGA 32 38 base pairs nucleic acid single linear cDNA NO NO 12 CTGGTTCGGCCCACCTCTGA AGGTTCCAGA ATCGATAG 38 35 base pairs nucleic acid singlelinear cDNA NO NO 13 GGAGACTTCC AAGGTCTTAG CTATCACTTA AGCAC 35 30 basepairs nucleic acid single linear cDNA NO 14 GCGCGCGTCG ACGAACACCCCCTCTTTGGC 30 30 base pairs nucleic acid single linear cDNA NO NO 15GCGCGCAAGC TTTCATAAGT GTGCGTGCTC 30 25 base pairs nucleic acid singlelinear cDNA NO NO 16 CCCGCGCGGC TTTTACATTA GGAGT 25 25 base pairsnucleic acid single linear cDNA NO NO 17 GCTGGCAAAC ATGCCCTCCT CATTG 2524 base pairs nucleic acid single linear cDNA NO NO 18 TGATGGCATGGACTGTGGTC ATGA 24 24 base pairs nucleic acid single linear cDNA NO NO19 TGATGGCATG GACTGTGGTC ATGA 24

What is claimed is:
 1. A DNA sequence encoding a protein or portionthereof, which inhibits vascular smooth muscle cell proliferation. 2.The DNA sequence of claim 1, comprising the nucleotide sequencesubstantially as shown in FIG.
 1. 3. The DNA sequence of claim 1,comprising the nucleotide sequence substantially as shown in FIG.
 3. 4.The DNA sequence of claim 1, comprising the nucleotide sequence fromabout 200 to about 1108 substantially as shown in FIG.
 1. 5. The DNAsequence of claim 1, comprising the nucleotide sequence from about 749to about 919 substantially as shown in FIG.
 1. 6. The messenger RNAtranscript of the DNA of claim
 1. 7. An isolated protein which inhibitsvascular smooth muscle cell growth.
 8. The protein of claim 7 having amolecular weight of from about 30 kDA to about 36 kDa.
 9. The protein ofclaim 7, comprising the amino acid sequence substantially as shown inFIG.
 1. 10. The protein of claim 7, comprising the amino acid sequencesubstantially as shown in FIG.
 3. 11. The protein of claim 7, comprisingthe amino acid sequence from about 1 to about 303 substantially as shownin FIG.
 1. 12. The protein of claim 7, further comprising glutathioneS-transferase.
 13. A vector containing the DNA sequence of claim
 1. 14.The vector of claim 13, wherein the nucleotide sequence is thenucleotide sequence of claim
 2. 15. The vector of claim 13, wherein thenucleotide sequence is the nucleotide sequence of claim
 3. 16. Thevector of claim 13, wherein the nucleotide sequence is the nucleotidesequence of claim
 4. 17. The vector of claim 13, wherein the nucleotidesequence is the nucleotide sequence of claim
 5. 18. A host celltransformed by vector of claim 13 containing the nucleotide sequencecoding for Gax protein.
 19. A host cell transformed by vector of claim14 containing the nucleotide sequence coding for Gax protein.
 20. A hostcell transformed by vector of claim 15 containing the nucleotidesequence coding for Gax protein.
 21. A host cell transformed by vectorof claim 16 containing the nucleotide sequence encoding the Gax protein.22. A host cell transformed by vector of claim 17 containing thenucleotide sequence encoding the Gax protein.
 23. A process for thepreparation of Gax protein comprising culturing the transformed host ofclaim 18 under conditions suitable for the expression of Gax protein andrecovering the Gax protein.
 24. A process for the preparation of aprotein which inhibits the proliferation of vascular smooth muscle cellscomprising culturing the transformed host of claim 19 under conditionssuitable for the expression of the protein and recovering the protein.25. A process for the preparation of a protein which inhibits theproliferation of vascular smooth muscle cells comprising culturing thetransformed host of claim 20 under conditions suitable for theexpression of the protein and recovering the protein.
 26. A process forthe preparation of a protein which inhibits the proliferation ofvascular smooth muscle cells comprising culturing the transformed hostof claim 21 under conditions suitable for the expression of the proteinand recovering the protein.
 27. The Gax protein made by the processselected from the processes of claim 23, claim 24, claim 25, and claim26.
 28. A method for inhibiting the proliferation of eukaryotic cells,comprising the following steps: a. providing nucleic acid sequenceencoding a protein, or portion thereof, which inhibits vascular smoothmuscle cell proliferation; and b. administering said nucleic acid orsaid protein to the cells.
 29. The method of claim 28 wherein thenucleic acid is DNA.
 30. The method of claim 28 wherein the nucleic acidis RNA.
 31. The method of claim 28 wherein the protein comprises theamino acid sequence from about 1 to 303 shown in FIG.
 1. 32. The methodof claim 28 wherein the cells are vascular smooth muscle cells.